Purpose: This study aimed to evaluate the utility of optical coherence tomography (OCT) in the quantification of eccentric fixation in amblyopic patients. Material and Methods: In this study, 14 amblyopic patients and 10 healthy volunteers were enrolled. Under non-mydriatic conditions, fixation tests were performed directly using a fixation ophthalmoscope and indirectly using spectral-domain OCT. For evaluations using OCT, the distance between the fovea and the fixation point, which was determined by a cross-sectional image, was measured. Results: On evaluations of healthy volunteers by OCT, the mean distance between the fixation point and the fovea was 80.4 ± 37.7 μm for the dominant eyes and 63.7 ± 36.4 μm for non-dominant eyes (p = 0.41). In amblyopic patients, on evaluation by OCT, the mean distance between the fixation point and the fovea was 193.8 ± 188.3 μm in amblyopic eyes and 83.5 ± 39.3 μm in paired fellow eyes (p = 0.02). Although OCT could detect eccentric fixation points in all the affected eyes of amblyopic patients, fixation ophthalmoscope was unable to quantify them in 2 of 14 affected eyes. Conclusions: Compared with a fixation ophthalmoscope, our method using OCT seems to be superior both in quantification and detection of eccentric fixation in amblyopic patients, without the need for mydriasis.

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